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The biological action of cellular depressants and stimulants I. *
The Biological Action of Cellular Depressants and Stimulants I* The Action of Ethyl Carbamate (Urethane) on the Endogenous Respiration and the Rate of Cell Division in Tetrahymena pyrifrmis By JOHN J. EILER, JOSEPH 2. KREZANOSKIt, and KWAN-HUA LEE The effect of urethane on the endogenous respiration and on the rate of cell multiplication has been studied in normal and in trained cells of Tetrabymena pyriformis with the view of obtaining information regarding the mode of action of narcotics.
of otherwise inhibitory concentrations of a drug suggested to us a possible means of putting to test the relationship suggested by Ormsbee and Fisher (6). Considerable support would be gained for the proposed gearing if, for example, organisms trained to grow in otherwise inhibitory concentrations of drug showed a concomitant decrease in sensitivity of a specific fraction of the respiration toward the depressing effects of the drug. Related to the question of the gearing between respiration and an endergonic process is the problem of whether the primary site of action of the drug is on respiration or on the endergonic process (3). A given set of results obtained in a training experiment, as just cited, could shed light on this vexing problem as well. In the experiments reported here, we have studied the effects of urethane (ethyl carbamate) on the rate of cell multiplication and on the endogenous respiration in trained and untrained cultures of Tetrahymena pyriformis.
HE CLASSICAL VIEW (1) that narcotics exert Ttheir characteristic biological effect through inhibition of cellular respiration persists at this time as one of several working hypotheses (2, 3). Despite the excellent studies by Quastel (4)and his associates, it has not been established that a depression of the respiratory activity in an oxygen-consuming cell is a primary, or even a necessary, event in the biological action of the narcotic or depressant drugs. In studies on several classes of unicellular organisms, Fisher and co-workers (5-8) have shown that narcotics cause almost complete cessation of cell division at concentrations which depress cellular respiration only moderately. I n studies on Tetruhymenu, Ormsbee and Fisher (6) showed that urethane caused almost complete inhibition of cell multiplication a t a concentration (0.1 M ) which inhibited the endogenous respiration only about 25 per cent. Further, the regression line relating the rate of the endogenous respiration to an extended range of drug concentrations was such as to suggest the existence of two parallel resiratory systems with the more drug-sensitive system being geared to the endergonic processes of cell multiplication. Numerous studies (9-11) on a variety of biological forms are compatible with the view that growth and other energy-requiring processes may be geared to a specific fraction of the total respiration. Indeed, recent studies (12) on rat liver mitochondria suggest alternate pathways of electron transport, only one of which is phosphorylative and work promoting. The successes which have been achieved (13, 14) in training organisms to grow in the presence
*
EXPERIMENTAL AND RESULTS Materials and Methods.-The W strain of Tetruhymem pyriformis used in this study was graciously
Received November 28, 1958, from the School of Pharmacy, University of California, San Francisco 22. A preliminary report of these results was presented in Fcderation Pvoc.. 15, No. 1, March, 1956. t Fellow of the American Foundation for Pharmaceutical Education. Present address: Medical College of Virginia, Richmond.
supplied by Prof. Daniel Mazia. Stock cultures were maintained in 300-1111. Erlenmeyer flasks containing 40 ml. of a medium consisting of 1.8% proteose-peptone (Difco) and 0.2% yeast extract (Difco). The cultures were maintained at 27' in t h e dark with weekly transfers. The sterility of both the stock and the experimental cultures was checked by microscopic observation and by plating on agar. Urethane was added to the medium as a sterile solution prior to inoculation. The appropriate concentration was sterilized by passage through a bacterial filter. The turbidity measurements were made with a Klett-Summerson photoelectric colorimeter, using the No. 42 filter to reduce the influence of the colored medium. All turbidity values are reported in Klett units. The procedure used for the counting of cells was essentially that of Hall, Johnson, and Loefer (15), using a Sedgewick-Rafter counting chamber (Scientific Glass Co., No. W4000) and a Whipple ocular micrometer. Cells were killed through addition of an aliquot of diluted formalin to yield a final concentration of 1% formaldehyde. When necessary, samples were diluted with a 0.24% solution of SOdium chloride. The total number of cells counted in
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each sample was between 500 and 1,000. Recently Scherbaum (16) has established that the error in this counting procedure is about 5%. The alkali-soluble biuret-detectable protein was determined on washed cell preparations using the procedure of Gornall, et al. (17). Cells were collected and washed with the above-mentioned sclution of sodium chloride through the use of centrifugation a t 2,500 r. p. m. (International Centrifuge, size 1) for five minutes. The biuret reagent was added directly to t h e cells washed free of medium and t h e absorbance was determined in a model B Beckman Spectrophotometer, using crystalline bovine albumin a s a standard. The values so obtained were the same as when t h e washed cells were first treated with trichloroacetic acid and the precipitated protein washed with diluted trichloroacetic acid prior to the addition of the biuret reagent. The simpler of the two procedures was used since all values are comparative and not absolute. Growth Studies.-All critical growth trials were carried out using 2,000-ml. Povitsky flasks containing 750 ml. of culture medium. The reasonably large surface-to-volume ratio thus obtained fostered rapid growth and the large volume permitted the frequent withdrawal of samples without marked changes in the surface-volume ratio of the culture. Further, in this fashion, the growth studies were conducted under the same conditions as were necessary t o obtain sufficient cells for the respiration studies. First, the effect of various concentrations of urethane on t h e rate of increase of cell mass was determined in order t o insure t h a t our strain of Tetrahymena behaved similarly t o the one used by Ormsbee and Fisher (6). The results of such growth trials testing the effect of six concentrations of urethane are presented in Fig. 1. In these trials each flask, containing 750 ml. either of the medium or the medium with the indicated concentration of urethane, was inoculated with 5.0 ml. of a suspension ( 5 X lobcells) from a stock culture in the rapid phase of growth. The flasks were incubated at 27" in the dark. Aliquots were withdrawn about every twelve hours for the determination of turbidity and cell protein. Only the results of the protein determinations are given in Fig. 1 since the Klett's units and the protein content were directly related throughout all growth trials with and without the several concentrations of urethane. The results, showing a graded effect of the several concentrations of the drug with essentially no growth at 0.11 M , are in good agreement with the findings of Ormsbee and Fisher (6). To test the possibility of training the protozoa to overcome, at least partially, the growth-inhibiting effects of urethane, the organisms were subcultured in Klett-Summerson tubes containing 5.0 ml. of either 2.0% proteose-peptone (Difco) or the same medium with urethane. The 0.1 ml. of inoculum was taken from a proteose-peptone culture in the stationary phase of growth. The tubes were incubated in the dark at 25'. Two-hundredths M urethane was selected as the concentration for initial exposure of the cells t o undergo training, since at this concentration growth is only slightly inhibited or even stimulated. Both normal cells and the cells undergoing training were subcultured every five days. The cells under training were transferred
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Fig. 1.-The effect of several concentrations of urethane on the growth of normal cultures: 0 = control; X = 0.03 M ; 0 = 0.056 M ; A = 0.07 M ; = 0.09 M ; A = 0.10 M, and = 0.11 M. The ordinate represents the experimental value times 100. twice into medium containing 0.02 M urethane and twice into medium containing 0.04 M drug. The normal or control cells were subcultured with the same frequency. To test the results of training, the time-dependent increases in turbidity of the normal cells in the presence and in the absence of 0.08 M urethane were compared with the increases of t h e drug-exposed cells in the presence of 0.04 M and 0.08 M urethane. The results presented in Fig. 2 indicate t h a t the cells trained in 0.04 M drug increased in mass at about the same rate in the presence of either 0.04 M or 0.08 M urethane as did the normal cells in the absence of the drug. The cells previously unexposed to the drug showed a greatly retarded rate of increase of mass in the presence of the 0.08 M urethane. Clearly, training under these conditions leads to an almost complete reduction in the sensitivity to the growth-inhibiting effects of 0.08 M urethane. The pronounced lag in growth observed in all trials in Fig. 2 is due to the use of mature cultures as inocula in the subculturing and in the testing, and is not due to the action of t h e drug. Similar studies indicated that concentrations above 0.08 M urethane could not be used for testing. When the training concentration was raised to 0.1 M only meager growth was observed for several subculturings, followed by complete failure of growth. It was necessary to alter the conditions for training t o provide sufficient uniform material for the later studies. All subsequent subculturing made use of 300-ml. Erlenmeyer flasks containing 40 ml. of medium with or without urethane. To permit more rapid growth than that indicated in Fig. 2, the medium consisted of 1.8 proteose-peptone and 0.2% yeast extract, as in the stock cultures. Trials testing the effectiveness of training under the new conditions were carried out using turbidity as the index of cell mass. The results of one set of trials are presented in Fig. 3. The cells undergoing training were brought through a series of concentra-
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Fig. 2.-The effect of urethane on the growth of test tube cultures of trained and untrained cells: = trained in 0.04 M urethane and 0 = control; tested in 0.04 M ; X = trained in 0.04 M urethane and tested in 0.08 M ; A = untrained and tested in 0.08 M.
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Fig. 3.-The effect of urethane on trained and untrained cells which had been cultured in Erlenmeyer flasks containing the standard medium. Normal cells, 0 = control; A = 0.07 M ; 0 = 0.08 M, and = 0.09 M. Trained cells, 0 = control; A = 0.07 M; rn = 0.08 &I, and e = 0.09 M.
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tions of urethane and, a t the time of the growth trials, had been subcultured in 0.08 Murethane every forty-eight hours for four times (two hundred sixteen hours). Control cells were subcultured in the plain medium with the same frequency. The growth trials were carried out in 300-ml. Erlenmeyer flasks fitted with a side arm suitable for insertion into the Klett-Summerson instrument. Forty ml. of medium or medium with urethane were inoculated with 0.5 ml. of suspension from either of the two filial subcultures.
The results given in Fig. 3 indicate that subculturing and testing under conditions which permit greater oxygenation (surface-volume ratio) and more rapid growth do not give rise to so great a decrease in sensitivity to the drug as was observed in the results presented in Fig. 2. All three test concentrations of urethane produced a proportional decrease in the rate of increase in cell mass. However, there remains a significant effect of training, since in each test concentration the drug exposed cells multiplied (turbidity) more rapidly than the previously unexposed cells. Similar studies demonstrated that shorter periods of training in 0.08 M urethane produced no measurable increase in drug resistance. Extension of the training period, with frequent subculturings, during fifteen days, or even six months, did not give better results than those presented in Fig. 3. In n o case did training permit growth in the presence of 0.1 M urethane. To observe the effect of training on increase in cell mass as judged both by turbidity and cell count, growth trials were conducted in Povitsky flasks. The 750 ml. of medium with or without urethane were inoculated with either trained or normal cells to yield an initial density of about 1,000 cells per ml. The trained cells had been subcultured in 0.08 M urethane every forty-eight hours for fifteen days. The normal cells were subcultured with the same frequency. Samples were withdrawn approximately every twelve hours during the incubation period. To conserve space, the results of the in5uence of the drug on the turbidity of the cultures are presented in Fig. 4 only as a regression line correlating turbidity with cell count. The cell count and Klett reading for every sample taken from both the trained and untrained cell cultures were used in the preparation of Fig. 4. While several factors undoubtedly contribute to the observed lack of linearity in the regression line, the principal factor relates t o dead cells and debris. As the cultures increase in age, the number of dead cells and the debris contribute to the Klett readings in a manner not reflected in the cell counts which included principally live cells. It is significant to point out that the results from both types of cells fit the curve equally well. This fact precludes the need t o present both cell count and turbidity data and suggests (18) that there is no great disparity in size between the normal and the trained cells. Closer attention was not given to the effect of training on cell size and morphology. The results of the effect of the drug on the rate of increase in cell count are presented in Fig. 5 as a semilog curve, wherein the time-dependent count divided by the initial count is plotted on a logarithmic scale against time. I t is clear from Fig. 5 that both the trained and the untrained cells multiplied at an almost identical rate in the absence of drug, while the trained cells multiplied more rapidly in each of the test concentrations of drug. Thus, training produced an increase in drug resistance as judged both by turbidity (cell mass) and cell count. The lack of linearity which develops a t about the fifth generation is related to oxygen want and will be considered more fully in the second paper in this series (19). The period of logarithmic growth, as is
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TABLEI.-THE EFFECTOF ETHYLCARBAMATE ON GENERATION TIMEOF NORMAL A N D TRAINED Telrahymena
THE
Cell Type
Molar Concn. of Urethane
Normal
0.00
Trained
0.07 0.08 0.09 0.00 0.07 0.08 0.09
Mean Generation Time" Time, Hr. Increase. % '
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3.4 4.4 5.0 5.6 3.4 4.0 4.3 4.7
29 47 65
.. 18 26 38
The mean generation times were calculated from the first-order rate constants estimated from the slopes of the curves in Fig. 5.
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Fig. 4.-Correlation of cell numbers with Klett readings. Open circles relate to normal cells; solid circles relate to trained cells. The values on the ordinate are t o be multiplied by 1.000 to yield the experimental value. customary, has been used t o calculate the mean generation time, as given in Table I, to provide quantitative estimates of the effect of training. The values for the per cent increase in mean generation time presented in Table I indicate that each of the test concentrations have about one-half the inhibitory effect on the trained cells as upon the untrained cells. Respiration Studies.-The effect of various concentrations of urethane on the rate of the endogenous respiration of normal and trained cells was measured at 27" essentially according t o the procedure of Ormsbee and Fisher, using the conventional Warburg technique. The drug-exposed cells had been subjected t o weekly subculturing in 0.08 M urethane for the period of approximately six months. To obtain sufficient cells, the final growth was conducted in Povitsky flasks. The normal cells were harvested from the i 5 0 ml. of medium at the end of forty-eight hours while the trained cells were harvested from the 750 ml. of urethane-containing medium a t the end of seventy-two hours. I n this
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Fig. 5.-The effect of several concentrations of urethane on trained and untrained cells. The timedependent cell count (PT)divided by the initial cell count (PO) is plotted on a logarithmic scale. Normal cells, 0 = control; A = 0.07 M ; 0 = 0.08, and 0 = 0.09 M . Trained cells, 0 = control; A = 0.07 M ; = 0.08 M , and = 0.09 M .
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way, approximately the same number of cells were obtained from each population. The cells were washed three times with 0.005 M phosphate buffer (pH 6.9) through the use of an angle head centrifuge. Each centrifugation was carried out for three minutes at 150 X g. The washed cells were suspended in 30 ml. of the phosphate buffer to yield a suspension containing about 2.5 mg. of cell protein per ml. (1.7 X lo6 normal cells per ml.). After about twenty minutes of time for preparation, the Warburg vessels were loaded with 1.0 ml. of the cell suspension, to which was added 0.5 ml. of the appropriate concentration of urethane in phosphate buffer, additional phosphate buffer was added to give a final volume of 2.3 ml. in the main compartment in all vessels. The center well was charged with alkali (0.2 ml. 20% KOH) and filter paper in the usual manner. After a ten-minute period for temperature equilibrium, the respiration trials were carried out
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with air as the gas phase and with shaking a t the rate of 130-134 cm. excursions per minute. It was established that the values reported were not limited by the rate of diffusion of oxygen. The uptake of oxygen was measured for a period of either sixty or ninety minutes, during which time the rate was essentially constant except in concentrations in excess of 0.2 M wherein the rate fell markedly and the effect of the drug was not reversible. The rate (pl. 02/hour/mg. protein) of the endogenous respiration of the normal cell amounted to 33, while that of the trained cell amounted to 34. These almost identical values are in good agreement with values in the literature reported on a dry weight basis (20, 21, 22) when allowance is made for the difference in the manner of expressing the values. Only one set of the many trials carried out is reported in the data presented in Fig. 6. Each point on the sets of curves in Fig. 6 represents the average of the results from two vessels from each of two cultures. Ten concentrations of urethane, ranging from 0.025 M to 0.2 M , were studied in addition to the non-urethane controls. The results presented in Fig. 6 are in the form of the “mass-action law” plot used by Ormsbee and Fisher: U is the rate of oxygen consumption in the presence of a given concentration of urethane; I is the difference between U and the respiration in the absence of urethane.’ As first pointed out by Ormsbee and Fisher, the effect of urethane on the rate of the endogenous respiration is best characterized by two intersecting straight lines. I n the case of both classes of cells, the lines intersect at a urethane concentration of about 0.11 M . In the results of Ormsbee and Fisher, the break in the curve was observed a t 0.1 M urethane. The difference in the sensitivity to the drug of the more sensitive respiration, as indicated
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in Fig. 6, is small. At 0.11 M urethane the endogenous respiration is depressed 26%. while the respiration of the trained cell is depressed 357& The differences are less a t lower concentrations. At the lowest concentration, the difference amounts t o about 4%. In any event, the trained cell is more, rather than less, sensitive to the respiratory depressing effects of urethane. This finding is in contrast to the effects on growth, wherein the trained cell is significantly less sensitive. Metabolism of Drug.-While these studies had little concern with the mechanism by which the increase in drug resistance took place, it was deemed necessary to exclude the possibility that the improved growth was due t o the circumstance that the trained cells metabolized urethane faster and thus caused a reduction in the relative concentration. Due t o the greater exposure of the cells to urethane in the growth trials, in contrast t o the respiration trials, an increased capacity to metabolize urethane would bias the growth results more than the respiration data. Accordingly, we tested the ability of the trained cell to metabolize urethane. An amount of urethane t o yield a final concentration of 0.08 M was added to each of four 750-ml. portions of medium and to each of two 750-ml. volumes of water. Two of the flasks containing medium were inoculated with trained cells and incubated for seventy-two hours, after which the cells were removed by centrifugation. One hundredmilliliter aliquots were then removed from each of the cell-free media and from each of the remaining vessels and extracted repeatedly with ethyl ether by a standardized procedure. The urethane in each of the ether extracts was crystallized with the use of petroleum ether, dried, and weighed. The averages of the duplicate weights from each set of aliquots were: 475 mg. from water; 475 mg. from the plain medium, and 480 mg. from the medium which had supported cell growth. The melting points of the several samples were in the range 4850”. The agreement in amounts recovered by the standardized procedure indicates that degradation of urethane is not significant and that it is not the mechanism by which training in growth takes place.
DISCUSSION
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Fig. 6.-The effect of urethane on the endogenous respiration of trained and untrained cells. The data are plotted according to the formulation of Ormsbee and Fisher (see text). According to Fisher ( l l ) , log ( U / I ) = log K - a log N , where K is the dissociation constant of a drug-enzyme complex, N is the concentration of drug, and a is the number of molecules of drug uniting with each active enzyme site.
The results presented in Figs. 1 and 6, pertaining to the control cells, ‘establish the fact that despite some differences in experimental conditions, the strain of Telrahymena used in these experiments responded to urethane similarly to the strain used by Ormsbee and Fisher (6). As in their results, the “mass-action law” plot of the results relating the rates of endogenous respiration to the concentration of urethane is best represented by two intersecting straight lines (Fig. 6). Likewise, the concentration of drug associated with the point of intersection is the same as that which inhibited cell multiplication almost completely. According t o them, the respiratory activity suggested by the line with the lesser slope is related in a specific fashion to the process of cell multiplication. The respiratory function represented by the greater slope was considered t o be associated with the vegetative activity of the cell. The argument was advanced by us (to present only one possibility) that support for their proposal would be obtained if cells trained t o grow in wethane
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showed an appropriate decrease in the sensitivity of the respiration to the depressing effect of the drug. The significance of the results would be enhanced if the change in sensitivity were confined t o that portion of the respiration suggested by the lesser slope. The results presented in Figs. 2, 3, and 5 demonstrate clearly that training in urethane promoted measurable increases in the resistance of the cultures t o the growth-inhibiting effect of the drug. However, the results obtained under conditions of poor oxygenation and slow growth (Fig. 2) stand in sharp contrast t o the results obtained under conditions of good aeration and more rapid growth. The cell trained in 0.04 M urethane under conditions of poor oxygenation developed a complete insensitivity to the growth-inhibiting effect of 0.08 M urethane. It is noteworthy that even with the degree of training established relative t o the effect of 0.08 M urethane, it was not possible to train the organisms to grow, even slowly, in 0.1 M urethane. The limited degree of training which we have achieved with Tetrahymena is in marked contrast t o the successes achieved by Hinshelwood (13)and co-workers working with bacteria. It is generally recognized that protozoa adapt t o the inhibitory effect of most drugs only slowly and not extensively (23). The results presented in Fig. 6 indicate definitely that neither the more sensitive respiration nor the total endogenous respiration showed a decrease in drug sensitivity as a result of training. Indeed, the adapted cells were slightly more sensitive to the respiratory effect of the drug. These findings indicate quite definitely that there is no change in respiration, reflected in the endogenous respiration, t h a t would account for the degree of training obtained in the growth studies. In a similar fashion, Preston and Eiler (24) could find no decrease in sensitivity t o the respiration depressing effect of pentobarbital in brain slices from rats rendered tolerant to the action of the drug. However, McCashland ( 2 5 ) recently observed an adaptive increase in the respiration, measured under the conditions of growth, in Tetrahymena trained t o resist the effects of potassium cyanide. The absence of a rigid coupling between respiration and cell multiplication, however, does not preclude a significant relation between the two processes. An important aspect of cell respiration, together with the anaerobic metabolism, is the supply of energy it provides for endergonic processes. The energy-rich phosphate compounds formed as a result of aerobic oxidations, and t o a lesser extent anaerobic oxidations, make possible growth and division of the cell. A coupling between respiration and growth based upon supply and demand for energy-rich compounds is a necessity in the life of aerobic organisms. A significant depression in respiration must be accompanied by a decline in endergonic processes unless compensated by an increase in the rate of the reactions which supply energy under anaerobic conditions. It is possible that just such a n increase in anaerobic metabolism may be basic to the improvement in growth observed in our experiments. The greater resistance to the growth-inhibiting effects of urethane established under the conditions of poor oxygenation and slow growth (Fig. 2) suggests that the principal change in the trained cell is
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the development of a more significant anaerobic metabolism. Ryley (21) has shown that Tetrahymena pyriformis is able to survive and maintain motility under anaerobic conditions. The anaerobic metabolism, at least of mammalian cells, is not nearly so sensitive to the inhibitory effects of narcotics as is the aerobic metabolism; indeed, the aerobic glycolysis of brain is increased by narcotics (4). An increase in anaerobic metabolism is a logical consequence of subculturing in relatively anaerobic conditions, especially when the aerobic metabolism has been depressed somewhat by urethane. The acceptance of a n adaptive change in the anaerobic metabolism carries with it the idea that growth and division are inhibited primarily through the action of the drug on respiration. Certainly, if the growth-inhibiting effects are, in part, reversed by the development of a n anaerobic metabolism, it is not reasonable that that fraction of the growth could have been inhibited by a direct effect of the drug on the growth process per se. It should be clear that the respiration whose inhibition has been considered to lead t o a n inhibition of growth is not the respiration which has been measured in these experiments; rather, it is the respiration which is measured under conditions in which cell division is taking place. The possible relation between the two respirations, relative t o growth, is not known at this time. Fortunately, such interrelations, together with the behavior of the anaerobic reactions, are susceptible to experimental study.
SUMMARY
1. The effects of urethane on the endogenous respiration and o n the rate of cell multiplication has been studied on trained a n d untrained cells of Tetrahymena pyriformis with the view of gaining some understanding of the mode of action of narcotic drugs. 2. T h e results of the action of the drug on the untrained cells are in good agreement with the earlier findings of Ormsbee and Fisher which suggested a significant relation between cell multiplication and respiration. 3. The cells from cultures which had been trained by repeated subculturing in the drug showed a higher rate of cell multiplication i n a given concentration of drug than did the untrained cells. The difference was most marked in cultures which have been trained under conditions of poor oxygenation. However, the endogenous respiration of the trained cells in a given concentration of urethane was no higher than t h a t of the untrained cells. 4. T h e results are considered i n terms of a possible mode of action of urethane.
REFERENCES (1) Verworn M “The Harvey Lectures” (1911-1912). J. B. Lippincot(Co.’,’ Philadelphia, Pa., 1912, p. 52. (2) Butler, T. C., J. Pharrnacol. Exfit!. TheraQ.. 98, 121 (1950).
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(3) Larabee, M. G . , Ramon, J. G., and Bielbring, E.. J . Cellular Corn$. Phy!jol.. 40, 461(1952). (4) Quastel, J. H., Neurochemistry,” Charles C Thomas, Springfield. Ill., 1955, p. 648. (5) Fisher, K. C., and Henry, R. J., J. Gen. Physiol.. 27,
469 (1944). (6) Ormshee, R. A,, and Fisher, K. C., ibid.. 27, 461 (1944). (7) Fisher, K. C., and Armstrong, F. H.. ibid., 30, 263 (1947). (8) Armstrong, F. H., and Fisher, K. C.. ibid., 30, 279 (1947). (9) Commoner, B., Qua!t. Res. B i d , 17 4F(1942). (10) Stern.. -1. R... and Fisher. K. C.. T i e Collecfana N e t . 17; 3(1912). (11) Fisher, K. C., Can. Med. Assoc. J . , 47, 414(1942). (12) Ernster, L., Jalliug. O., Low, H., and Lindberg, O., Ezfifl.Cell. Research, Suppl., 3 , 124(1955). (13).Hinsbe!=~ood,C. N., The Chemical Kinetics of the Bacterral Cell, Clarendon Press, Oxford, England, 1946, p. 95.
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(I!) Schnitzer. R. J., and Grunberg, E.. “Drug Resistance of Microorganisms,” Academic Press, New York, N. Y., 1957, p. 87. (15) Hall R. P. Johnson D. F. and Loefer, J. B., Trans. Am. M i c r o s ~ o p .Sdc., 54, 218(19353. (16) Scherbaum.. 0.. . Acfa Pathol. Microbiol. Scand.., 40.. 7 (i957j. (17) Gornall, A. G., Bardowill, C. J.. and David, M. M.. J. Biol. Chem. 177 751(1949). (18) Mckashiand, B. W., J . Profomol.,2,97(1955). (19) Eiler, J. J.. Krezaaoski, J. Z., and Lee, K.-H.. to be published. (20) Eichel, H. J., and Rotb, J. S., B i d . Bull., 104, 351 (IYSB).
(21) Ryley, J. F., Biochem. J., 5 2 , 480, 483(1952). (22) Ormshee, R. A,, B i d . Bull., 82, 423(1942). (23) Goodwin, L. G.. and Rollo, I. M., Protozoa,” Ed. by Hunter, S. H., and Lwoff. H., Academic Press, New York. 1955, p . 247. (24) Preston, J. E., and Eiler, J . J., to be published. (25) McCasbland, B. W., J . Profozool.,3, 131(1956).
Dialkylaminoethyl Esters of 4-Nitro- and 4-Amino-3 -hydroxy-2-naphthoic Acids* By GEORGE M. SIEGERt, WILLIAM M. ZIEGLERt, DAVID X. KLEIN, and HERMAN SOKOL The 2-diethylaminoethylester of 3-hydroxy-2naphthoic acid was nitrated in acetic acid solution to yield the corresponding ringsubstitufed 4-nitro derivative. Catalytic hydrogenation of the nitro compound g a v e 2-diethylaminoethyl 4-amino-3-hydroxy-2naphthoate hydrochloride. The anesthetic properties of these nitro and amino derivatives have been studied and compared with unsubstituted 3-hydroxy-2-naphthoic acid esters of the same series. (1) the synthesis of a number of dialkylaminoalkyl esters and amides derived from 3-hydroxy- and 3-alkoxy-2naphthoic acids and some of their physiological properties were described. In continuation of these earlier studies, it was deemed worthwhiIe to prepare ring-substituted nitro and amino derivatives of the 2-diethylaminoethyl ester of 3-hydroxy-2-naphthoic acid in order to determine whether or not the anesthetic activity could be enhanced with relation to the unsubstituted ester. The rationale supporting the possibility for an increased anesthetic effect lies in the fact that the proposed compounds could be considered naphthoic acid analogs of ring-substituted local anesthetics, such as procaine (2-diethylaminoethyl 4-amino-1-benzoate) and Naphthocaine (2-diethylaminoethyl 4-amino-1-naphthoate) and their related 4-nitro compounds (3, 4). Since it has been demonstrated in several series of the
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N A PREVIOUS PUBLICATION
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Received September 24, 1958, from the Research Laboratories of the Heyden Newport Chemical Corp., Garfield, N. J. t Present address: Lederle Laboratories, American Cyanamid Co.. Pearl River, N. Y. The authors wish t o thank Mr. Joseph Marrus and Dr. E. S. Echague for the physiological tests and Mr. Hilding Johnson and members of his staff for the microanalyses. The authors also wish t o acknowledge the valuable cooperation and assistance offered by Drs. Ralph N. Lulek, Robert H. Barth, Neil E. Rigler, Cyril D. Wilson, and John E. Snow in carrying out these studies.
!ocal anesthetics that the introduction of second ring substituents, such as amino or alkoxyl groups, sometimes increases the activity (3-91, there seemed to be a stdiciently good reason for preparing a ring-amino derivative of 2-diethylaminoethyl 3-hydroxy-2-naphthoate for testing as a local anesthetic. Synthesis of the proposed ring-substituted amino derivative of 2-diethylaminoethyl 3-hydroxy-2-naphthoate was accomplished by nitrating the hydrochloride of this ester (I) in glacial acetic acid under the same conditions described by Gradenwitz (2) for the preparation of methyl 4-nitro-3-hydroxy-2-naphthoate(V) and subsequent reduction of the nitro derivative (11) to the corresponding amino compound (111) by catalytic hydrogenation.
I1 - R = NO2 111 - R = NH2
Designation of the nitro and amino groups as ring substituents in the 4-position is consistent with the work of Gradenwitz (2) who obtained the 4-nitro ring derivative of the methyl ester of 3-hpdroxy-2-naphthoic acid (V) under conditions similar to those used for nitration of the diethylaminoethyl ester. These structures were esta blished further by hydrolyzing both the nitrated methyl and 2-diethylaminoethyl esters (V and 11)
of otherwise inhibitory concentrations of a drug suggested to us a possible means of putting to test the relationship suggested by Ormsbee and Fisher (6). Considerable support would be gained for the proposed gearing if, for example, organisms trained to grow in otherwise inhibitory concentrations of drug showed a concomitant decrease in sensitivity of a specific fraction of the respiration toward the depressing effects of the drug. Related to the question of the gearing between respiration and an endergonic process is the problem of whether the primary site of action of the drug is on respiration or on the endergonic process (3). A given set of results obtained in a training experiment, as just cited, could shed light on this vexing problem as well. In the experiments reported here, we have studied the effects of urethane (ethyl carbamate) on the rate of cell multiplication and on the endogenous respiration in trained and untrained cultures of Tetrahymena pyriformis.
HE CLASSICAL VIEW (1) that narcotics exert Ttheir characteristic biological effect through inhibition of cellular respiration persists at this time as one of several working hypotheses (2, 3). Despite the excellent studies by Quastel (4)and his associates, it has not been established that a depression of the respiratory activity in an oxygen-consuming cell is a primary, or even a necessary, event in the biological action of the narcotic or depressant drugs. In studies on several classes of unicellular organisms, Fisher and co-workers (5-8) have shown that narcotics cause almost complete cessation of cell division at concentrations which depress cellular respiration only moderately. I n studies on Tetruhymenu, Ormsbee and Fisher (6) showed that urethane caused almost complete inhibition of cell multiplication a t a concentration (0.1 M ) which inhibited the endogenous respiration only about 25 per cent. Further, the regression line relating the rate of the endogenous respiration to an extended range of drug concentrations was such as to suggest the existence of two parallel resiratory systems with the more drug-sensitive system being geared to the endergonic processes of cell multiplication. Numerous studies (9-11) on a variety of biological forms are compatible with the view that growth and other energy-requiring processes may be geared to a specific fraction of the total respiration. Indeed, recent studies (12) on rat liver mitochondria suggest alternate pathways of electron transport, only one of which is phosphorylative and work promoting. The successes which have been achieved (13, 14) in training organisms to grow in the presence
*
EXPERIMENTAL AND RESULTS Materials and Methods.-The W strain of Tetruhymem pyriformis used in this study was graciously
Received November 28, 1958, from the School of Pharmacy, University of California, San Francisco 22. A preliminary report of these results was presented in Fcderation Pvoc.. 15, No. 1, March, 1956. t Fellow of the American Foundation for Pharmaceutical Education. Present address: Medical College of Virginia, Richmond.
supplied by Prof. Daniel Mazia. Stock cultures were maintained in 300-1111. Erlenmeyer flasks containing 40 ml. of a medium consisting of 1.8% proteose-peptone (Difco) and 0.2% yeast extract (Difco). The cultures were maintained at 27' in t h e dark with weekly transfers. The sterility of both the stock and the experimental cultures was checked by microscopic observation and by plating on agar. Urethane was added to the medium as a sterile solution prior to inoculation. The appropriate concentration was sterilized by passage through a bacterial filter. The turbidity measurements were made with a Klett-Summerson photoelectric colorimeter, using the No. 42 filter to reduce the influence of the colored medium. All turbidity values are reported in Klett units. The procedure used for the counting of cells was essentially that of Hall, Johnson, and Loefer (15), using a Sedgewick-Rafter counting chamber (Scientific Glass Co., No. W4000) and a Whipple ocular micrometer. Cells were killed through addition of an aliquot of diluted formalin to yield a final concentration of 1% formaldehyde. When necessary, samples were diluted with a 0.24% solution of SOdium chloride. The total number of cells counted in
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each sample was between 500 and 1,000. Recently Scherbaum (16) has established that the error in this counting procedure is about 5%. The alkali-soluble biuret-detectable protein was determined on washed cell preparations using the procedure of Gornall, et al. (17). Cells were collected and washed with the above-mentioned sclution of sodium chloride through the use of centrifugation a t 2,500 r. p. m. (International Centrifuge, size 1) for five minutes. The biuret reagent was added directly to t h e cells washed free of medium and t h e absorbance was determined in a model B Beckman Spectrophotometer, using crystalline bovine albumin a s a standard. The values so obtained were the same as when t h e washed cells were first treated with trichloroacetic acid and the precipitated protein washed with diluted trichloroacetic acid prior to the addition of the biuret reagent. The simpler of the two procedures was used since all values are comparative and not absolute. Growth Studies.-All critical growth trials were carried out using 2,000-ml. Povitsky flasks containing 750 ml. of culture medium. The reasonably large surface-to-volume ratio thus obtained fostered rapid growth and the large volume permitted the frequent withdrawal of samples without marked changes in the surface-volume ratio of the culture. Further, in this fashion, the growth studies were conducted under the same conditions as were necessary t o obtain sufficient cells for the respiration studies. First, the effect of various concentrations of urethane on t h e rate of increase of cell mass was determined in order t o insure t h a t our strain of Tetrahymena behaved similarly t o the one used by Ormsbee and Fisher (6). The results of such growth trials testing the effect of six concentrations of urethane are presented in Fig. 1. In these trials each flask, containing 750 ml. either of the medium or the medium with the indicated concentration of urethane, was inoculated with 5.0 ml. of a suspension ( 5 X lobcells) from a stock culture in the rapid phase of growth. The flasks were incubated at 27" in the dark. Aliquots were withdrawn about every twelve hours for the determination of turbidity and cell protein. Only the results of the protein determinations are given in Fig. 1 since the Klett's units and the protein content were directly related throughout all growth trials with and without the several concentrations of urethane. The results, showing a graded effect of the several concentrations of the drug with essentially no growth at 0.11 M , are in good agreement with the findings of Ormsbee and Fisher (6). To test the possibility of training the protozoa to overcome, at least partially, the growth-inhibiting effects of urethane, the organisms were subcultured in Klett-Summerson tubes containing 5.0 ml. of either 2.0% proteose-peptone (Difco) or the same medium with urethane. The 0.1 ml. of inoculum was taken from a proteose-peptone culture in the stationary phase of growth. The tubes were incubated in the dark at 25'. Two-hundredths M urethane was selected as the concentration for initial exposure of the cells t o undergo training, since at this concentration growth is only slightly inhibited or even stimulated. Both normal cells and the cells undergoing training were subcultured every five days. The cells under training were transferred
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Fig. 1.-The effect of several concentrations of urethane on the growth of normal cultures: 0 = control; X = 0.03 M ; 0 = 0.056 M ; A = 0.07 M ; = 0.09 M ; A = 0.10 M, and = 0.11 M. The ordinate represents the experimental value times 100. twice into medium containing 0.02 M urethane and twice into medium containing 0.04 M drug. The normal or control cells were subcultured with the same frequency. To test the results of training, the time-dependent increases in turbidity of the normal cells in the presence and in the absence of 0.08 M urethane were compared with the increases of t h e drug-exposed cells in the presence of 0.04 M and 0.08 M urethane. The results presented in Fig. 2 indicate t h a t the cells trained in 0.04 M drug increased in mass at about the same rate in the presence of either 0.04 M or 0.08 M urethane as did the normal cells in the absence of the drug. The cells previously unexposed to the drug showed a greatly retarded rate of increase of mass in the presence of the 0.08 M urethane. Clearly, training under these conditions leads to an almost complete reduction in the sensitivity to the growth-inhibiting effects of 0.08 M urethane. The pronounced lag in growth observed in all trials in Fig. 2 is due to the use of mature cultures as inocula in the subculturing and in the testing, and is not due to the action of t h e drug. Similar studies indicated that concentrations above 0.08 M urethane could not be used for testing. When the training concentration was raised to 0.1 M only meager growth was observed for several subculturings, followed by complete failure of growth. It was necessary to alter the conditions for training t o provide sufficient uniform material for the later studies. All subsequent subculturing made use of 300-ml. Erlenmeyer flasks containing 40 ml. of medium with or without urethane. To permit more rapid growth than that indicated in Fig. 2, the medium consisted of 1.8 proteose-peptone and 0.2% yeast extract, as in the stock cultures. Trials testing the effectiveness of training under the new conditions were carried out using turbidity as the index of cell mass. The results of one set of trials are presented in Fig. 3. The cells undergoing training were brought through a series of concentra-
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Fig. 2.-The effect of urethane on the growth of test tube cultures of trained and untrained cells: = trained in 0.04 M urethane and 0 = control; tested in 0.04 M ; X = trained in 0.04 M urethane and tested in 0.08 M ; A = untrained and tested in 0.08 M.
'-1 160 IS0 140
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Fig. 3.-The effect of urethane on trained and untrained cells which had been cultured in Erlenmeyer flasks containing the standard medium. Normal cells, 0 = control; A = 0.07 M ; 0 = 0.08 M, and = 0.09 M. Trained cells, 0 = control; A = 0.07 M; rn = 0.08 &I, and e = 0.09 M.
+
tions of urethane and, a t the time of the growth trials, had been subcultured in 0.08 Murethane every forty-eight hours for four times (two hundred sixteen hours). Control cells were subcultured in the plain medium with the same frequency. The growth trials were carried out in 300-ml. Erlenmeyer flasks fitted with a side arm suitable for insertion into the Klett-Summerson instrument. Forty ml. of medium or medium with urethane were inoculated with 0.5 ml. of suspension from either of the two filial subcultures.
The results given in Fig. 3 indicate that subculturing and testing under conditions which permit greater oxygenation (surface-volume ratio) and more rapid growth do not give rise to so great a decrease in sensitivity to the drug as was observed in the results presented in Fig. 2. All three test concentrations of urethane produced a proportional decrease in the rate of increase in cell mass. However, there remains a significant effect of training, since in each test concentration the drug exposed cells multiplied (turbidity) more rapidly than the previously unexposed cells. Similar studies demonstrated that shorter periods of training in 0.08 M urethane produced no measurable increase in drug resistance. Extension of the training period, with frequent subculturings, during fifteen days, or even six months, did not give better results than those presented in Fig. 3. In n o case did training permit growth in the presence of 0.1 M urethane. To observe the effect of training on increase in cell mass as judged both by turbidity and cell count, growth trials were conducted in Povitsky flasks. The 750 ml. of medium with or without urethane were inoculated with either trained or normal cells to yield an initial density of about 1,000 cells per ml. The trained cells had been subcultured in 0.08 M urethane every forty-eight hours for fifteen days. The normal cells were subcultured with the same frequency. Samples were withdrawn approximately every twelve hours during the incubation period. To conserve space, the results of the in5uence of the drug on the turbidity of the cultures are presented in Fig. 4 only as a regression line correlating turbidity with cell count. The cell count and Klett reading for every sample taken from both the trained and untrained cell cultures were used in the preparation of Fig. 4. While several factors undoubtedly contribute to the observed lack of linearity in the regression line, the principal factor relates t o dead cells and debris. As the cultures increase in age, the number of dead cells and the debris contribute to the Klett readings in a manner not reflected in the cell counts which included principally live cells. It is significant to point out that the results from both types of cells fit the curve equally well. This fact precludes the need t o present both cell count and turbidity data and suggests (18) that there is no great disparity in size between the normal and the trained cells. Closer attention was not given to the effect of training on cell size and morphology. The results of the effect of the drug on the rate of increase in cell count are presented in Fig. 5 as a semilog curve, wherein the time-dependent count divided by the initial count is plotted on a logarithmic scale against time. I t is clear from Fig. 5 that both the trained and the untrained cells multiplied at an almost identical rate in the absence of drug, while the trained cells multiplied more rapidly in each of the test concentrations of drug. Thus, training produced an increase in drug resistance as judged both by turbidity (cell mass) and cell count. The lack of linearity which develops a t about the fifth generation is related to oxygen want and will be considered more fully in the second paper in this series (19). The period of logarithmic growth, as is
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SCIENTIFIC EDITION
TABLEI.-THE EFFECTOF ETHYLCARBAMATE ON GENERATION TIMEOF NORMAL A N D TRAINED Telrahymena
THE
Cell Type
Molar Concn. of Urethane
Normal
0.00
Trained
0.07 0.08 0.09 0.00 0.07 0.08 0.09
Mean Generation Time" Time, Hr. Increase. % '
..
3.4 4.4 5.0 5.6 3.4 4.0 4.3 4.7
29 47 65
.. 18 26 38
The mean generation times were calculated from the first-order rate constants estimated from the slopes of the curves in Fig. 5.
240,
5 200 x220
.ooc
i
3;-, 1 8 0 3 160 J
140
-
120
-
100
-
Fig. 4.-Correlation of cell numbers with Klett readings. Open circles relate to normal cells; solid circles relate to trained cells. The values on the ordinate are t o be multiplied by 1.000 to yield the experimental value. customary, has been used t o calculate the mean generation time, as given in Table I, to provide quantitative estimates of the effect of training. The values for the per cent increase in mean generation time presented in Table I indicate that each of the test concentrations have about one-half the inhibitory effect on the trained cells as upon the untrained cells. Respiration Studies.-The effect of various concentrations of urethane on the rate of the endogenous respiration of normal and trained cells was measured at 27" essentially according t o the procedure of Ormsbee and Fisher, using the conventional Warburg technique. The drug-exposed cells had been subjected t o weekly subculturing in 0.08 M urethane for the period of approximately six months. To obtain sufficient cells, the final growth was conducted in Povitsky flasks. The normal cells were harvested from the i 5 0 ml. of medium at the end of forty-eight hours while the trained cells were harvested from the 750 ml. of urethane-containing medium a t the end of seventy-two hours. I n this
,0
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-1
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TIME IHOUrlSI
Fig. 5.-The effect of several concentrations of urethane on trained and untrained cells. The timedependent cell count (PT)divided by the initial cell count (PO) is plotted on a logarithmic scale. Normal cells, 0 = control; A = 0.07 M ; 0 = 0.08, and 0 = 0.09 M . Trained cells, 0 = control; A = 0.07 M ; = 0.08 M , and = 0.09 M .
+
=
way, approximately the same number of cells were obtained from each population. The cells were washed three times with 0.005 M phosphate buffer (pH 6.9) through the use of an angle head centrifuge. Each centrifugation was carried out for three minutes at 150 X g. The washed cells were suspended in 30 ml. of the phosphate buffer to yield a suspension containing about 2.5 mg. of cell protein per ml. (1.7 X lo6 normal cells per ml.). After about twenty minutes of time for preparation, the Warburg vessels were loaded with 1.0 ml. of the cell suspension, to which was added 0.5 ml. of the appropriate concentration of urethane in phosphate buffer, additional phosphate buffer was added to give a final volume of 2.3 ml. in the main compartment in all vessels. The center well was charged with alkali (0.2 ml. 20% KOH) and filter paper in the usual manner. After a ten-minute period for temperature equilibrium, the respiration trials were carried out
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with air as the gas phase and with shaking a t the rate of 130-134 cm. excursions per minute. It was established that the values reported were not limited by the rate of diffusion of oxygen. The uptake of oxygen was measured for a period of either sixty or ninety minutes, during which time the rate was essentially constant except in concentrations in excess of 0.2 M wherein the rate fell markedly and the effect of the drug was not reversible. The rate (pl. 02/hour/mg. protein) of the endogenous respiration of the normal cell amounted to 33, while that of the trained cell amounted to 34. These almost identical values are in good agreement with values in the literature reported on a dry weight basis (20, 21, 22) when allowance is made for the difference in the manner of expressing the values. Only one set of the many trials carried out is reported in the data presented in Fig. 6. Each point on the sets of curves in Fig. 6 represents the average of the results from two vessels from each of two cultures. Ten concentrations of urethane, ranging from 0.025 M to 0.2 M , were studied in addition to the non-urethane controls. The results presented in Fig. 6 are in the form of the “mass-action law” plot used by Ormsbee and Fisher: U is the rate of oxygen consumption in the presence of a given concentration of urethane; I is the difference between U and the respiration in the absence of urethane.’ As first pointed out by Ormsbee and Fisher, the effect of urethane on the rate of the endogenous respiration is best characterized by two intersecting straight lines. I n the case of both classes of cells, the lines intersect at a urethane concentration of about 0.11 M . In the results of Ormsbee and Fisher, the break in the curve was observed a t 0.1 M urethane. The difference in the sensitivity to the drug of the more sensitive respiration, as indicated
35 1
I
4
in Fig. 6, is small. At 0.11 M urethane the endogenous respiration is depressed 26%. while the respiration of the trained cell is depressed 357& The differences are less a t lower concentrations. At the lowest concentration, the difference amounts t o about 4%. In any event, the trained cell is more, rather than less, sensitive to the respiratory depressing effects of urethane. This finding is in contrast to the effects on growth, wherein the trained cell is significantly less sensitive. Metabolism of Drug.-While these studies had little concern with the mechanism by which the increase in drug resistance took place, it was deemed necessary to exclude the possibility that the improved growth was due t o the circumstance that the trained cells metabolized urethane faster and thus caused a reduction in the relative concentration. Due t o the greater exposure of the cells to urethane in the growth trials, in contrast t o the respiration trials, an increased capacity to metabolize urethane would bias the growth results more than the respiration data. Accordingly, we tested the ability of the trained cell to metabolize urethane. An amount of urethane t o yield a final concentration of 0.08 M was added to each of four 750-ml. portions of medium and to each of two 750-ml. volumes of water. Two of the flasks containing medium were inoculated with trained cells and incubated for seventy-two hours, after which the cells were removed by centrifugation. One hundredmilliliter aliquots were then removed from each of the cell-free media and from each of the remaining vessels and extracted repeatedly with ethyl ether by a standardized procedure. The urethane in each of the ether extracts was crystallized with the use of petroleum ether, dried, and weighed. The averages of the duplicate weights from each set of aliquots were: 475 mg. from water; 475 mg. from the plain medium, and 480 mg. from the medium which had supported cell growth. The melting points of the several samples were in the range 4850”. The agreement in amounts recovered by the standardized procedure indicates that degradation of urethane is not significant and that it is not the mechanism by which training in growth takes place.
DISCUSSION
- a 04
06
OB L O G 1O’N
10
12
14
Fig. 6.-The effect of urethane on the endogenous respiration of trained and untrained cells. The data are plotted according to the formulation of Ormsbee and Fisher (see text). According to Fisher ( l l ) , log ( U / I ) = log K - a log N , where K is the dissociation constant of a drug-enzyme complex, N is the concentration of drug, and a is the number of molecules of drug uniting with each active enzyme site.
The results presented in Figs. 1 and 6, pertaining to the control cells, ‘establish the fact that despite some differences in experimental conditions, the strain of Telrahymena used in these experiments responded to urethane similarly to the strain used by Ormsbee and Fisher (6). As in their results, the “mass-action law” plot of the results relating the rates of endogenous respiration to the concentration of urethane is best represented by two intersecting straight lines (Fig. 6). Likewise, the concentration of drug associated with the point of intersection is the same as that which inhibited cell multiplication almost completely. According t o them, the respiratory activity suggested by the line with the lesser slope is related in a specific fashion to the process of cell multiplication. The respiratory function represented by the greater slope was considered t o be associated with the vegetative activity of the cell. The argument was advanced by us (to present only one possibility) that support for their proposal would be obtained if cells trained t o grow in wethane
May 1959
SCIENTIFIC EDITION
showed an appropriate decrease in the sensitivity of the respiration to the depressing effect of the drug. The significance of the results would be enhanced if the change in sensitivity were confined t o that portion of the respiration suggested by the lesser slope. The results presented in Figs. 2, 3, and 5 demonstrate clearly that training in urethane promoted measurable increases in the resistance of the cultures t o the growth-inhibiting effect of the drug. However, the results obtained under conditions of poor oxygenation and slow growth (Fig. 2) stand in sharp contrast t o the results obtained under conditions of good aeration and more rapid growth. The cell trained in 0.04 M urethane under conditions of poor oxygenation developed a complete insensitivity to the growth-inhibiting effect of 0.08 M urethane. It is noteworthy that even with the degree of training established relative t o the effect of 0.08 M urethane, it was not possible to train the organisms to grow, even slowly, in 0.1 M urethane. The limited degree of training which we have achieved with Tetrahymena is in marked contrast t o the successes achieved by Hinshelwood (13)and co-workers working with bacteria. It is generally recognized that protozoa adapt t o the inhibitory effect of most drugs only slowly and not extensively (23). The results presented in Fig. 6 indicate definitely that neither the more sensitive respiration nor the total endogenous respiration showed a decrease in drug sensitivity as a result of training. Indeed, the adapted cells were slightly more sensitive to the respiratory effect of the drug. These findings indicate quite definitely that there is no change in respiration, reflected in the endogenous respiration, t h a t would account for the degree of training obtained in the growth studies. In a similar fashion, Preston and Eiler (24) could find no decrease in sensitivity t o the respiration depressing effect of pentobarbital in brain slices from rats rendered tolerant to the action of the drug. However, McCashland ( 2 5 ) recently observed an adaptive increase in the respiration, measured under the conditions of growth, in Tetrahymena trained t o resist the effects of potassium cyanide. The absence of a rigid coupling between respiration and cell multiplication, however, does not preclude a significant relation between the two processes. An important aspect of cell respiration, together with the anaerobic metabolism, is the supply of energy it provides for endergonic processes. The energy-rich phosphate compounds formed as a result of aerobic oxidations, and t o a lesser extent anaerobic oxidations, make possible growth and division of the cell. A coupling between respiration and growth based upon supply and demand for energy-rich compounds is a necessity in the life of aerobic organisms. A significant depression in respiration must be accompanied by a decline in endergonic processes unless compensated by an increase in the rate of the reactions which supply energy under anaerobic conditions. It is possible that just such a n increase in anaerobic metabolism may be basic to the improvement in growth observed in our experiments. The greater resistance to the growth-inhibiting effects of urethane established under the conditions of poor oxygenation and slow growth (Fig. 2) suggests that the principal change in the trained cell is
295
the development of a more significant anaerobic metabolism. Ryley (21) has shown that Tetrahymena pyriformis is able to survive and maintain motility under anaerobic conditions. The anaerobic metabolism, at least of mammalian cells, is not nearly so sensitive to the inhibitory effects of narcotics as is the aerobic metabolism; indeed, the aerobic glycolysis of brain is increased by narcotics (4). An increase in anaerobic metabolism is a logical consequence of subculturing in relatively anaerobic conditions, especially when the aerobic metabolism has been depressed somewhat by urethane. The acceptance of a n adaptive change in the anaerobic metabolism carries with it the idea that growth and division are inhibited primarily through the action of the drug on respiration. Certainly, if the growth-inhibiting effects are, in part, reversed by the development of a n anaerobic metabolism, it is not reasonable that that fraction of the growth could have been inhibited by a direct effect of the drug on the growth process per se. It should be clear that the respiration whose inhibition has been considered to lead t o a n inhibition of growth is not the respiration which has been measured in these experiments; rather, it is the respiration which is measured under conditions in which cell division is taking place. The possible relation between the two respirations, relative t o growth, is not known at this time. Fortunately, such interrelations, together with the behavior of the anaerobic reactions, are susceptible to experimental study.
SUMMARY
1. The effects of urethane on the endogenous respiration and o n the rate of cell multiplication has been studied on trained a n d untrained cells of Tetrahymena pyriformis with the view of gaining some understanding of the mode of action of narcotic drugs. 2. T h e results of the action of the drug on the untrained cells are in good agreement with the earlier findings of Ormsbee and Fisher which suggested a significant relation between cell multiplication and respiration. 3. The cells from cultures which had been trained by repeated subculturing in the drug showed a higher rate of cell multiplication i n a given concentration of drug than did the untrained cells. The difference was most marked in cultures which have been trained under conditions of poor oxygenation. However, the endogenous respiration of the trained cells in a given concentration of urethane was no higher than t h a t of the untrained cells. 4. T h e results are considered i n terms of a possible mode of action of urethane.
REFERENCES (1) Verworn M “The Harvey Lectures” (1911-1912). J. B. Lippincot(Co.’,’ Philadelphia, Pa., 1912, p. 52. (2) Butler, T. C., J. Pharrnacol. Exfit!. TheraQ.. 98, 121 (1950).
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(3) Larabee, M. G . , Ramon, J. G., and Bielbring, E.. J . Cellular Corn$. Phy!jol.. 40, 461(1952). (4) Quastel, J. H., Neurochemistry,” Charles C Thomas, Springfield. Ill., 1955, p. 648. (5) Fisher, K. C., and Henry, R. J., J. Gen. Physiol.. 27,
469 (1944). (6) Ormshee, R. A,, and Fisher, K. C., ibid.. 27, 461 (1944). (7) Fisher, K. C., and Armstrong, F. H.. ibid., 30, 263 (1947). (8) Armstrong, F. H., and Fisher, K. C.. ibid., 30, 279 (1947). (9) Commoner, B., Qua!t. Res. B i d , 17 4F(1942). (10) Stern.. -1. R... and Fisher. K. C.. T i e Collecfana N e t . 17; 3(1912). (11) Fisher, K. C., Can. Med. Assoc. J . , 47, 414(1942). (12) Ernster, L., Jalliug. O., Low, H., and Lindberg, O., Ezfifl.Cell. Research, Suppl., 3 , 124(1955). (13).Hinsbe!=~ood,C. N., The Chemical Kinetics of the Bacterral Cell, Clarendon Press, Oxford, England, 1946, p. 95.
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(I!) Schnitzer. R. J., and Grunberg, E.. “Drug Resistance of Microorganisms,” Academic Press, New York, N. Y., 1957, p. 87. (15) Hall R. P. Johnson D. F. and Loefer, J. B., Trans. Am. M i c r o s ~ o p .Sdc., 54, 218(19353. (16) Scherbaum.. 0.. . Acfa Pathol. Microbiol. Scand.., 40.. 7 (i957j. (17) Gornall, A. G., Bardowill, C. J.. and David, M. M.. J. Biol. Chem. 177 751(1949). (18) Mckashiand, B. W., J . Profomol.,2,97(1955). (19) Eiler, J. J.. Krezaaoski, J. Z., and Lee, K.-H.. to be published. (20) Eichel, H. J., and Rotb, J. S., B i d . Bull., 104, 351 (IYSB).
(21) Ryley, J. F., Biochem. J., 5 2 , 480, 483(1952). (22) Ormshee, R. A,, B i d . Bull., 82, 423(1942). (23) Goodwin, L. G.. and Rollo, I. M., Protozoa,” Ed. by Hunter, S. H., and Lwoff. H., Academic Press, New York. 1955, p . 247. (24) Preston, J. E., and Eiler, J . J., to be published. (25) McCasbland, B. W., J . Profozool.,3, 131(1956).
Dialkylaminoethyl Esters of 4-Nitro- and 4-Amino-3 -hydroxy-2-naphthoic Acids* By GEORGE M. SIEGERt, WILLIAM M. ZIEGLERt, DAVID X. KLEIN, and HERMAN SOKOL The 2-diethylaminoethylester of 3-hydroxy-2naphthoic acid was nitrated in acetic acid solution to yield the corresponding ringsubstitufed 4-nitro derivative. Catalytic hydrogenation of the nitro compound g a v e 2-diethylaminoethyl 4-amino-3-hydroxy-2naphthoate hydrochloride. The anesthetic properties of these nitro and amino derivatives have been studied and compared with unsubstituted 3-hydroxy-2-naphthoic acid esters of the same series. (1) the synthesis of a number of dialkylaminoalkyl esters and amides derived from 3-hydroxy- and 3-alkoxy-2naphthoic acids and some of their physiological properties were described. In continuation of these earlier studies, it was deemed worthwhiIe to prepare ring-substituted nitro and amino derivatives of the 2-diethylaminoethyl ester of 3-hydroxy-2-naphthoic acid in order to determine whether or not the anesthetic activity could be enhanced with relation to the unsubstituted ester. The rationale supporting the possibility for an increased anesthetic effect lies in the fact that the proposed compounds could be considered naphthoic acid analogs of ring-substituted local anesthetics, such as procaine (2-diethylaminoethyl 4-amino-1-benzoate) and Naphthocaine (2-diethylaminoethyl 4-amino-1-naphthoate) and their related 4-nitro compounds (3, 4). Since it has been demonstrated in several series of the
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N A PREVIOUS PUBLICATION
*
Received September 24, 1958, from the Research Laboratories of the Heyden Newport Chemical Corp., Garfield, N. J. t Present address: Lederle Laboratories, American Cyanamid Co.. Pearl River, N. Y. The authors wish t o thank Mr. Joseph Marrus and Dr. E. S. Echague for the physiological tests and Mr. Hilding Johnson and members of his staff for the microanalyses. The authors also wish t o acknowledge the valuable cooperation and assistance offered by Drs. Ralph N. Lulek, Robert H. Barth, Neil E. Rigler, Cyril D. Wilson, and John E. Snow in carrying out these studies.
!ocal anesthetics that the introduction of second ring substituents, such as amino or alkoxyl groups, sometimes increases the activity (3-91, there seemed to be a stdiciently good reason for preparing a ring-amino derivative of 2-diethylaminoethyl 3-hydroxy-2-naphthoate for testing as a local anesthetic. Synthesis of the proposed ring-substituted amino derivative of 2-diethylaminoethyl 3-hydroxy-2-naphthoate was accomplished by nitrating the hydrochloride of this ester (I) in glacial acetic acid under the same conditions described by Gradenwitz (2) for the preparation of methyl 4-nitro-3-hydroxy-2-naphthoate(V) and subsequent reduction of the nitro derivative (11) to the corresponding amino compound (111) by catalytic hydrogenation.
I1 - R = NO2 111 - R = NH2
Designation of the nitro and amino groups as ring substituents in the 4-position is consistent with the work of Gradenwitz (2) who obtained the 4-nitro ring derivative of the methyl ester of 3-hpdroxy-2-naphthoic acid (V) under conditions similar to those used for nitration of the diethylaminoethyl ester. These structures were esta blished further by hydrolyzing both the nitrated methyl and 2-diethylaminoethyl esters (V and 11)